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36 6
JMed Genet 1996;33:36-41
Haplotype analysis in prenatal diagnosis and
carrier identification of Salla disease
Johanna Schleutker, Pertti Sistonen, Pertti Aula
Department of
Medical Genetics,
Institute of
Biomedicine,
University of Turku,
Kiinamyllynkatu 10,
FIN-20520 Turku,
Finland
J Schleutker
P Aula
Finnish Red Cross
Blood Transfusion
Service, Helsinki,
Finland
P Sistonen
Correspondence
to:
Dr Schleutker.
Received 24 July 1995
Accepted for publication
7 September 1995
Abstract
considerably higher frequency of 0 013 has
Salla disease (SD) is an autosomal re- been estimated for the high incidence area.7
cessive disorder in which free sialic acid The gene frequencies in other populations are
(N-acetyl neuraminic acid) accumulates not known, but rare sporadic families have
in lysosomes. A specific transport mech- been described.89 Lysosomal accumulation of
anism for acidic monosaccharides on the free sialic acid also leads to a phenotypically
lysosomal membrane has recently been different clinical entity, the infantile type of free
described, but the molecular deficiency sialic acid storage disease (ISSD). Prenatal
causing SD is still unknown. We have pre- diagnosis of SD and ISSD has so far been
viously mapped the SD gene to 6ql4-ql5 based on the increased intracellular free sialic
by means of genetic linkage analysis and acid content in cultured amniotic fluid cells,"
restricted the positive chromosomal area or in CVS samples in ISSD.'2-'4
to less than 100 kb with linkage disWe have assigned the gene for SD to 6q1 4-15
equilibrium mapping. The two best allelic by linkage analyses in Finnish families.7 '5 The
association markers have now retro- locus homogeneity of different clinical phenospectively been used in five prenatal ana- types of free sialic acid storage diseases was
lyses originally studied with sialic acid recently shown in patients from different ethnic
assays in chorionic villus specimens. In backgrounds. 16 A founder effect derived linkage
four cases an unaffected fetus was pre- disequilibrium in the Finnish population is eviddicted with a probability level of more than ent between the disease locus and the closest
94%, which was in concordance with the markers AFMa336zf9 and AFMb28 lwg9.
biochemical data. One fetus was predicted Eighty-five percent of Finnish SD patients are
to be affected with over 96% probability, homozygous for the 1-3 haplotype of these two
as was shown by free sialic acid assays in microsatellite markers, and there is only one
a CVS sample and in fetal tissues after family (out of a total of 50 families) with two
termination of the pregnancy. Risk cal- affected sisters who lack the 1-3 haplotype and
culations incorporating disequilibrium are homozygous for the 1-8 haplotype.
were also used to predict the carrier status
Here we assess the value of linkage disin members of six families with previous equilibrium in prenatal diagnosis of SD by
SD cases, and also in a few cases with no calculating retrospectively the risks of being
known family history of SD. DNA marker affected in five at risk pregnancies, which were
based analysis thus provides a reliable originally studied by free sialic acid assay in
method for risk estimations in prenatal uncultured CVS samples. We also applied
cases and for carrier identification of SD. haplotype analysis to estimate the probabilities
of carrier status in family members of SD
(7Med Genet 1996;33:36-41)
patients.
In one of the prenatal cases an affected child
Key words: Salla disease; prenatal diagnosis.
was predicted, whereas in four instances SD
was excluded with probability levels ranging
Salla disease (SD), also known as Finnish or from 96 to 100%. These results correlated
adult type free sialic acid storage disorder well with the original sialic acid data. Carrier
identification could be performed in all cases
(MIM 269920), is caused by accumulation with
a high probability level. Haplotype based
of NANA (N-acetylneuraminic acid) in the
calculation
can now be applied to identify
risk
lysosomes.' A specific transport mechanism
in the subpopulation
carriers
of
SD
particularly
for acidic monosaccharides on the lysosomal
in
Finland.
of
the
risk
area
high
membrane has been described and a defect in
the putative transport protein of NANA is the
likely cause of the disease.23 The diagnosis
of SD is based on early onset psychomotor Materials and methods
retardation associated with ataxia and some
other neurological abnormalities.4 The progression of the symptoms is relatively slow
and SD patients have a near normal life
expectancy.56 SD is inherited as an autosomal
recessive trait with exceptional enrichment in
the Finnish population, particularly in the
north-eastern part of the country, the Salla
region. A gene frequency of 0-006 has been
calculated for the whole of Finland, whereas a
PRENATAL DIAGNOSIS FAMILIES
Prenatal diagnosis had been performed in at
risk pregnancies by free sialic acid assay on
uncultured first trimester CVS samples in four
families (fig 1) with an earlier child affected
with Salla disease. Sialic acid was assayed with
an HPLC method (M Renlund, personal communication). In four pregnancies the sialic acid
content was within the control level, the pregnancies went to term, and healthy infants were
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37
Haplotype analysis in prenatal diagnosis and camrer identification of Salla disease
2
1
2
1 7
32
110146
32 68
1 6
37
1 i
3 7
1 8
3 6
34/
3
*0
1 4
36
1 1 76
33 27
37
3:
3
2
<3
1
2
33 33
1 7
3 3I
3 6 3 7
77
36
7 1 7 7 1 7
33 36 36
I I I I ND
33 33
Figure 1 Pedigrees of prenatal diagnosis families. The
corresponding haplotypes are given below each subject.
Risk was calculated for those subjects marked with an
arrow. The blackened symbols indicate a patient with
Salla disease, hatched symbols indicate a heterozygote.
2
1
2
1
1 2
1 7
3 6
3 7
17
33
5/
2
6/
8/
9/
72 12 15 75
15
35 69 3335 35
43
27 1 1 1 7 1 1
67 33 37 33
11 75
22
93
33 35
4
3
1
2
7
8
ND
1 7
36
17
36
27
69
3
71
63
4
9
71
68
33
14
17
39
27 12
36 36
^o3
6
11 17
3836
8/
14
41 1147
333339
17
ND ND
72
66
19/0
12 17
69 39
21
jl
1 1
3 3
77
5*6
1 1
3 3
DNA SAMPLES
7
1
2
El}
V
1 7 1 2
3 6
3 6
I 1 7 1
3 3 63
2
3
4
27
17
63
37
12
36
17
37
1
21
6 3
7/
9 1 5 7
63 66
Figure 2 Pedigrees of the families for carrier identification.* denotes
marker allele. Results offamily 7 are only presented in the text.
one
CARRIER IDENTIFICATION FAMILIES
Twenty-two healthy members from five families
in which SD segregated requested estimation
of the probability of being a carrier of SD.
Eight of them were sibs of an SD patient. The
pedigrees of the families are shown in fig 2. In
family 2 carrier identification was requested by
a 25 year old pregnant woman (6) whose sister
had a daughter affected with SD. The husband
also originated from the high gene frequency
area and, in addition, he may be related to
another SD family. Family 3 with second cousins (5 and 12) affected with SD originates from
eastern Finland, outside the high risk area. Four
family members (indicated in fig 2) requested
carrier identification.
In two additional families (6 and 7) haplotype
analysis was performed for diagnostic purposes.
The newborn baby in family 6 had no signs of
SD nor any other abnormalities and the urinary
free sialic acid was within normal limits. The
parents had refused prenatal studies during the
pregnancy. In family 7 the haplotype analysis
was performed on a 9 month old male infant
whose clinical and sialic acid studies had led
to a strong suspicion of SD. He was hypotonic
and ataxic. MRI of the central nervous system
showed evidence of demyelination and the
amount of free sialic acid in urine was above
normal limits.
2/
6
10
2
4X 15.
77 59 1 7
4*6 6 6 3 6
11 11 71 11 11
36 36 36 33 33 33
27 17
9969
5
1
I11 7 1
36
delivered. Normal development and the absence of free sialic acid in urine have confirmed
the prenatal prediction in these children. In
one case (family 3) the raised free sialic acid
level in a CVS sample indicated that the fetus
was affected with SD and the pregnancy was
terminated. An increased level of free sialic
acid and the presence of enlarged vacuoles
in several tissues of the fetus confirmed the
diagnosis of SD.
Aliquots of CVS samples were used retrospectively for DNA extraction and haplotype
analysis using the two most closely linked
markers AFMa336zf9 and AFMb28 lwg9.
DNA samples from family members were studied simultaneously with the fetal samples. Prediction of the fetal status was based on linkage
analysis as described below.
1 1
3 3
|7/
I 1
3 3
a new
mutation in
DNA was extracted from venous blood in accordance with standard protocols and from
second trimester villus biopsy specimens, which
were originally taken for the determination of
free sialic acid." In one case the DNA was
extracted from a paraffin block using a modification of the method described by Dubeau
et al.'7 Deparaffinisation and rehydration was
carried out with 200 VI xylene, 600 gl 70%
ethanol (twice), followed by short incubation
at 55°C. After incubation a mixture of 75 ,l
H20, 10 l lysate buffer, 5 pl 10% Tween, and
10 gl proteinase K was added and the sample
was incubated overnight. The enzyme was inactivated at 95°C for 10 minutes and 3-5 ,l of
the solution was used in PCR.
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Schleutker, Sistonen, Aula
38
Table 1 Finnish population frequencies of AFMa336zf9-AFMb281wg9 haplotypes in normal and SD chromosomes
adjusted to 0-9870 for normal chromosomes and to 0-0130 for SD chromosomes according to the local SD gene frequency
of 0 013
SD chromosomes
Normal chromosomes
Haplotype
No
Frequency
Haplotype
No (%)
1-3
1-5
1-7
1-8
2-1
2-2
2-6
5-8
7-3
66
1
1
3
1
1
1
1
2
N(9)
77 (100)
1-2
1-3
1-6
1-7
2-3
2-5
2-6
2-7
2-8
2-9
3-1
3-2
3-6
3-7
4-3
4-6
5-5
5-6
5-8
6-3
6-7
6-8
7-1
7-2
7-3
7-4
7-6
7-7
7-8
7-9
8-2
8-3
8-6
8-7
9-3
9-6
10-2
2
1
2
2
3
1
10
2
1
2
2
1
2
1
2
2
1
2
1
2
3
1
2
4
16
1
11
6
1
1
1
2
1
1
2
1
2
0-020143
0-010071
0-020143
0-020143
0-030214
0-010071
0-100714
0-020143
0-010071
0-020143
0-020143
0 010071
0-020143
0-010071
0-020143
0-020143
0-010071
0-020143
0-010071
0-020143
0-030214
0-019971
0-020143
0-040286
0-161143
0-010071
0-110786
0-060429
0-010071
0-010071
0-010071
0-020143
0-010071
0-010071
0-020143
0-010071
0-020143
N (37)
98
0-9870
MICROSATELLITE DNA MARKERS AND DETECTION
OF POLYMORPHISMS
Two markers used in this study, AFMa336zf9
and AFMb281wg9, were provided by Genethon Resource Center. These markers had
earlier been shown to have the most significant
linkage disequilibrium in the Finnish SD families.'6 PCR based detection of microsatellite
polymorphisms was carried out as previously
described.7"'
RISK CALCULATIONS
Risk was computed
(86-0)
(1-25)
(1-25)
(3-90)
(1-25)
(1-25)
(1-25)
(1-25)
(2-60)
Frequency
0-011143
0-000169
0-000169
0-000506
0-000169
0-000169
0-000169
0-000169
0-000338
0-0130
Results
The segregation of alleles in the 10 families at
the two marker loci, AFMa336zf9 and
AFMb28 1wg9, previously shown to be closely
linked to the SD gene are shown in figs 1 and
2. Of the possible 180 haplotype combinations,
37 were observed in the normal chromosomes
while only nine were present in the SD chromosomes (table 1). The results of the risk
calculations with two marker haplotypes at
different values of 0 are shown in table 2 for
the prenatal cases and in table 3 for the carrier
identifications.
using the MLINK program
of the LINKAGE package.'8 Haplotype frequencies were counted in the Finnish Salla RISK CALCULATIONS IN PRENATAL CASES
families previously used for linkage analysis7 In families 1 and 2 the risk calculation gave a
and were combined with those included in the probability level of > 96-04% for an unaffected
risk calculations only. Altogether they consisted heterozygous fetus. In family 4 the first fetus
of 98 normal and 77 SD chromosomes. A three (4) was predicted to be homozygous for the
locus haplotype for each of the two marker normal gene, since the probability of SD hetloci and the Salla locus was constructed erozygosity was < 3 92%, and the risk of being
assuming a locus order of SD-AFMa336zf9- affected was below 0 04%. The second fetus
AFMb281wg9 with zero recombination be- was predicted to be a healthy heterozygote
tween the marker loci, as observed.'6 The three with a probability of ) 96-08%. In family 3 an
locus haplotype frequencies were included in affected fetus was predicted (homozygote for
the analysis under the assumption that the 1-3) with a probability level of 96&04%. All
observed linkage disequilibrium is valid and the results of the risk calculations are concordant with the prenatal studies performed
were adjusted to an estimated local SD population frequency of 0-013 (table 1). All the during pregnancy by sialic acid assay.
alleles observed (two for SD, 10 for AFMa336zf9, and nine for AFMb281wg9) were
included in the haplotypes which then con- CARRIER ANALYSIS
stituted a total of 180 possible haplotype com- Carrier detection was based on the high predictive value of the risk haplotype 1-3, which
binations.
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39
Haplotype analysis in prenatal diagnosis and carier identification of Salla disease
camrer
Table 2 Risks of
status (heterozygous 112) or affected (homozygous 212)
disease families studied as prenatal diagnosis cases (fig 1)
Risk (%) at given 0
phenotypes in four Finnish Salla
Family
Subject
Salla genotype
0 000
0-005
0-010
0-015
0-020
Predicted phenotype
1
1
1
1
2
3
4
97-98
0
97-98
0
100
0
0
100
0
0
100
0
97 50
0
97 00
0
99 00
0 50
1 00
99 00
1 00
0 00
99 01
0 50
97-01
0
96-03
0
98-01
099
1-98
98-01
1-98
0 01
98-02
099
96-53
0
95-06
0
97 03
1-48
2-96
97-02
2-98
0-02
97 05
1-48
96-04
0
94-10
0
96-07
1-96
3-92
96-04
3-92
0-04
96-08
1-96
Healthy SD carrier
Healthy non-carrier
Healthy SD carrier
2
1/2
1/2
1/2
2/2
1/2
2/2
1/2
2/2
1/2
2/2
1/2
2/2
3
4
4
4
4
5
Healthy SD carrier
Affected with SD
Healthy non-carrier
Healthy SD carrier
Table 3 Risks of carrier status (heterozygous 112) or affected (homozygous 2/2) phenotypes in six Finnish Salla disease
families studied as carier diagnosis cases (fig 2)
Risk (%) at given 0
Family
Subject
1
1
2
2
2
3
5
5
6
7
2
2
3
3
3
3
3
3
4
4
4
5
5
5
5
5
6
8
9
15
18
19
20
21
22
1
2
5
3
4
5
6
7
4
Salla genotype
0 000
0-005
0-010
0-015
0-020
1/2
1/2
1/2
1/2
1/2
2/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
2/2
0
100
0
100
0
0
100
0
100
0
0
100
0
0
100
0
100
0
0
100
100
0
100
0
1 00
99-50
0
99 50
0 50
0
99 50
0 50
99 50
0 50
0
99 50
0 50
0 50
99 50
1 00
99 50
1 00
0
99 50
99-00
0 50
99 01
0 50
1-98
99 00
0
99 00
0 99
0
99 00
1 00
99 00
1 00
0
99 00
0.99
2-96
98-50
0
98-50
1-48
0
98-50
1-50
98-50
1-50
0
98-50
1-48
1-48
98-50
2-96
98-50
2-96
0
98-50
97-00
1-50
97 05
1-48
98-00
0
98-88
1-96
0
98-00
2-00
98-00
2-00
0
98-00
1-96
1-96
98-00
3-92
98-00
3-92
0
98-00
96-00
2-00
96-08
1-96
has so far been found only on one normal
chromosome in the Finnish population. In at
risk families, subjects with high risk values
> 96-08% were considered to be carriers of the
SD gene and those with risk values s3-92%
were identified as non-carriers. In family 5
a new mutation at the marker locus
AFMb281wg9 was detected either in subject
3 or 9, in whom either allele 4 or 5 must have
been mutated. However, this had no effect on
the risk calculations. Subjects without a family
history of SD (spouses of family members) had
carrier risk levels of 0%. The use of an SD
gene frequency of 0-006 had no effect on the
risk calculation. With lower 0 values of 0 0050-000, the corresponding risk estimations were
.t99-01% for carriers and <1% for non-carriers.
The risk was also calculated for four newborn
infants, at the request of the parents, to exclude
the disease. In family 2 the baby's (7) haplotype
yielded a 0% risk of being affected and the
carrier probability was s 1 96%, depending on
the chosen 0. The free sialic acid in the baby's
urine was within the normal range. The child
is now 6 months old and has shown no evidence
of developmental delay or any other signs of
SD. In family 3 the DNA of the twin girls (21)
and (22) was extracted from the placentas after
birth, and their haplotypes gave risk levels
s, 1 96% of being a carrier. No urine tests have
0-99
99 00
1-98
99 00
1-98
0
99 00
98-00
1 00
98-02
099
3-92
Predicted phenotype
Non-carrier
Carrier
Non-carrier
Carrier
Non-carrier
Carrier
Non-carrier
Carrier
Non-carrier
Non-carrier
Carrier
Non-carrier
Non-carrier
Carrier
Non-carrier
Carrier
Non-carrier
Non-carrier
Carrier
Carrier
Non-carrier
Carrier
been performed. The girls are now 14 months
of age and have developed normally. In family
6 the baby's (4) urine was examined shortly
after birth and normal levels of free sialic acid
were detected, suggesting a healthy child. DNA
analysis predicted him to be an SD heterozygote, with a probability of 96 08%,
compared to the risk of being affected of
< 1-96%. The boy is now 16 months of age
and has reached the normal milestones of development.
In family 7 the healthy mother was found to
be homozygous for the 1-3 haplotype, representing so far the only 1-3 haplotype in a
normal, non-SD chromosome. At the time of
the DNA analysis the diagnosis of her son,
the first child of unrelated parents, was still
equivocal but SD was strongly suspected on
the basis of his clinical findings. His subsequent
clinical course, repeated free sialic acid assays
of urine, and findings of demyelination of the
central nervous system on MRI has confirmed
the diagnosis of SD. The results of the haplotype analysis with 1-3 homozygosity are thus
fully consistent with the phenotypic findings.
However, if an unknown phenotype was assumed for the baby (as in a prenatal situation),
his risk of being an SD carrier would be
> 49-98% or >,41-08% and the risk of being
affected ,16-87% or r8-01%, with an SD
gene frequency of 0-013 or 0-006, respectively.
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40
Schleutker, Sistonen, Aula
Discussion
Previous experience with the present families
indicates that the determination of the level
of free sialic acid (NANA) from uncultured
chorionic villus samples is an accurate and
reliable method for prenatal diagnosis of SD
(M Renlund, personal communication). One
of the five fetuses in the present study material
had been found to be affected on the basis of
raised free sialic acid levels in the uncultured
chorionic villus sample. The pregnancy was
terminated and the diagnosis was confirmed
by EM and free sialic acid assays of fetal tissues.
Prenatal diagnosis of SD had been attempted
previously with cultured amniocytes by free
sialic acid and free/total sialic acid ratio determination. " However, cultured amniotic
fluid cells are not fully appropriate because the
increase in NANA in SD is only moderate and
the effect of the culture conditions is unknown.
In ISSD, the other phenotypic manifestation
of lysosomal sialic acid storage, the more pronounced rise of intracellular free sialic acid has
provided an even more straightforward approach for prenatal diagnosis.2'-4
The present study shows that as a result of
the assignment of the SD locus to 6q, linkage
analysis is an alternative method for prenatal
diagnosis. The strong linkage disequilibrium
observed in the Finnish SD families with
microsatellite markers AFMa336zf9 and
AFMb28lwg9'6 substantially adds to the
power of linkage analysis. No recombinations
have yet been detected between these two
markers and the SD locus. The risk haplotype
1-3 has been detected in 91% of parental
Finnish SD chromosomes but only once in a
normal chromosome, the mother of family 7 in
the present study. The haplotype frequencies,
together with the segregation of the disease in
the SD families, determine the risk which is
largely independent of the gene frequency given
strong linkage disequilibrium. The present case
is a good example of this. The correctness of
the risk calculation depends on the accuracy
of the estimation of haplotype frequency, primarily of that of the high association haplotype
in normal chromosomes.
The retrospective risk calculations correctly
predicted the phenotype of the fetus in all
the families. In the other recessive diseases
enriched in the Finnish population, prenatal
diagnosis using linkage disequilibrium has previously successfully been used in diastrophic
dyplasia,'9 infantile ceroid lipofuscinosis
(CLN1),20 and cartilage-hair hypoplasia.2' In
these diseases the degree of allelic associations
and the level of risk probabilities have been
approximately within the same range as in SD.
In non-Finnish families conventional linkage
analysis can now be used for prenatal diagnosis
in both SD and ISSD since no locus heterogeneity was detected in our previous linkage
studies of different phenotypes of free sialic
acid storage diseases.'6
A possible source of error is mutation of
microsatellites, as could be seen in family 5 in
our carrier studies. So far this is the only mutation event detected with markers AFMa336zf9
and AFMb281wg9 from about 500 chromo-
somes analysed. The estimated average mutation rate of microsatellites, 1 0-', is still
relatively low22 and data from various studies
suggest that microsatellites amplify accurately
enough to be suitable for diagnostic studies.
Our calculations were also carried out assuming
a mutation frequency of 10-' for the marker
AFMb281wg9 but this had no effect on the
risks. A possibility remains that the only detected 1-3 non-SD haplotype in family 7 results
from a new mutation either in AFMa336zf9 or
AFMb28 lwg9. However, no direct evidence
for this was detected when the extended haplotypes of the parents of the 1-3 homozygous
mother were analysed.
In the past, carrier identification in SD was
not possible. For subjects with no previous
history of SD and not originating from the
high risk area of Salla, a risk estimation was
calculated using both gene frequencies, 0-006
and 0-013. The gene frequency estimation of
0-006 was based on the annual incidence of
about two SD cases per 60000 births.7 For
those with no family history of SD but originating from the high risk area, a disease gene
frequency of 0-013 was used. This was based
on the carrier frequency estimation of 1:40 in
this area.2' However, the different gene frequencies had no significant effect on the calculated risks in these cases.
As mentioned above, the different estimates
of the SD gene frequency have little effect on
the risk of probabilities in the SD families
because their risks are based on segregation
and linkage. However, in a case of a person
from the general Finnish population, the risk
is based on the frequencies of the SD gene and
the 1-3 risk haplotype. We can now estimate
that the probability for any Finnish person
carrying the 1-3 risk haplotype of being an SD
carrier is approximately 33% or 51% depending
on the SD gene frequency of 0-006 or 0-013.
The corresponding figures for a 1-3 homo44% or 50% of being a
zygote would be
carrier and
10% or 25% of being affected.
The probability for a subject without the 1-3
haplotype of being an SD carrier can be estimated from the haplotype frequencies in normal and SD chromosomes (table 1). The risk
associated with all other haplotypes present in
both normal and SD chromosomes is very low,
ranging from 0-1 to 1-5%. Accordingly, the
rare haplotypes, such as 1-8 which has so far
been found in SD chromosomes only, would
indicate a high risk for heterozygosity but this
is probably because of the limited number of
chromosomes studied so far. These estimates
are substantially more accurate than the predictions based on the gene frequencies alone.
Haplotype analysis can therefore be used in
clinical practice to predict the risk of SD. A
couple who both lacked the 1-3 risk haplotype
would have a low risk of bearing an affected
child whereas a couple with both parents having
the high risk haplotype would run a considerable risk of having a child with SD. The
haplotype analysis will, however, mostly benefit
the family members of the SD patients who
wish to know their carrier status. As shown by
-
-
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41
Haplotype analysis in prenatal diagnosis and camrer identification of Salla disease
the present results, haplotype analysis can give
reliable predictions in most cases.
Cloning of the SD gene and identification of
the mutation causing the disease will eventually
replace the haplotype analysis in carrier detection and in prenatal diagnosis, at least in
the Finnish population. The observed linkage
disequilibrium strongly suggests that there is
only one major mutation causing the disease
in this founder population, as is the case with
aspartylglucosaminuria and very likely also with
the other genetic diseases enriched in the Finnish population.24 The presence of only one
or a few mutations would facilitate diagnostic
studies and even provide the possibility for
carrier screening in the general population.
The cooperation and positive attitude of all the SD families is
greatly appreciated. We thank Pirkko Jalava for her assistance
in the laboratory. This study was financially supported by
the Maud Kuistila Foundation, Finland and the Academy of
Finland.
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Haplotype analysis in prenatal diagnosis and
carrier identification of Salla disease.
J Schleutker, P Sistonen and P Aula
J Med Genet 1996 33: 36-41
doi: 10.1136/jmg.33.1.36
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